Multiple Myeloma Cells Alter Adipogenesis, Increase Senescence-Related and Inflammatory Gene Transcript Expression, and Alter Metabolism in Preadipocytes

Abstract

Within the bone marrow microenvironment, mesenchymal stromal cells (MSCs) are an essential precursor to bone marrow adipocytes and osteoblasts. The balance between this progenitor pool and mature cells (adipocytes and osteoblasts) is often skewed by disease and aging. In multiple myeloma (MM), a cancer of the plasma cell that predominantly grows within the bone marrow, as well as other cancers, MSCs, preadipocytes, and adipocytes have been shown to directly support tumor cell survival and proliferation. Increasing evidence supports the idea that MM-associated MSCs are distinct from healthy MSCs, and their gene expression profiles may be predictive of myeloma patient outcomes. Here we directly investigate how MM cells affect the differentiation capacity and gene expression profiles of preadipocytes and bone marrow MSCs. Our studies reveal that MM.1S cells cause a marked decrease in lipid accumulation in differentiating 3T3-L1 cells. Also, MM.1S cells or MM.1S-conditioned media altered gene expression profiles of both 3T3-L1 and mouse bone marrow MSCs. 3T3-L1 cells exposed to MM.1S cells before adipogenic differentiation displayed gene expression changes leading to significantly altered pathways involved in steroid biosynthesis, the cell cycle, and metabolism (oxidative phosphorylation and glycolysis) after adipogenesis. MM.1S cells induced a marked increase in 3T3-L1 expression of MM-supportive genes including Il-6 and Cxcl12 (SDF1), which was confirmed in mouse MSCs by qRT-PCR, suggesting a forward-feedback mechanism. In vitro experiments revealed that indirect MM exposure prior to differentiation drives a senescent-like phenotype in differentiating MSCs, and this trend was confirmed in MM-associated MSCs compared to MSCs from normal donors. In direct co-culture, human mesenchymal stem cells (hMSCs) exposed to MM.1S, RPMI-8226, and OPM-2 prior to and during differentiation, exhibited different levels of lipid accumulation as well as secreted cytokines. Combined, our results suggest that MM cells can inhibit adipogenic differentiation while stimulating expression of the senescence associated secretory phenotype (SASP) and other pro-myeloma molecules. This study provides insight into a novel way in which MM cells manipulate their microenvironment by altering the expression of supportive cytokines and skewing the cellular diversity of the marrow.

Keywords: adipocytes; bone marrow; mesenchymal stromal cells (MSCs); microarray; myeloma; preadipocytes; senescence.

Figure 1

Expression of genes encoding key metabolic proteins in myeloma patient mesenchymal stromal cells (MSCs). Expression of transcripts involved in PPAR signaling Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (A), in MSCs derived from normal patient bone marrow (NBM) or myeloma patients (MM), graphically demonstrated using Morpheus software (The Broad Institute). Groups of down and upregulated genes can roughly be seen in the top and bottom boxed regions. Reduced gene expression of FADS1 (B) and FADS2 (C) and increased expression of ANGPTL4 (D). Analysis of publicly available data from Corre et al. 2007, Leukemia.

Figure 2

Expression of genes encoding key adipogenic proteins in myeloma patient mesenchymal stromal cells (MSCs). Expression of transcripts included in the Hallmark Adipogenesis Gene Set, in MSCs derived from normal patient bone marrow (NBM) or myeloma patients (MM). Graphically demonstrated using Morpheus software (The Broad Institute). Analysis of publicly available data from Corre et al. 2007, Leukemia.

Figure 3

Mesenchymal stromal cells (MSCs) derived from myeloma patients exhibit impaired adipogenic differentiation. Experimental design of MSC differentiation experiment (A). MSCs differentiated into adipocytes for 21 days from normal donor bone marrow (NBM-BMAds), (B), or myeloma patient bone marrow (MM-BMAds, (C) for 21 days; images taken at 20X, scale bars = 250 µm. PPARG (D), CEBPA (E), and FABP4 (F) expression are decreased at the end of the adipogenic differentiation period in adipocytes derived from myeloma patient MSCs (MM-BMAds), relative to normal donor controls (NBM-BMAds) (n=3); *p < 0.05.

Figure 4

Mouse bone marrow mesenchymal stem cells (BM-MSCs) exposed to multiple myeloma (MM) cells via indirect co-culture in vitro exhibit reduced adipogenesis. Experimental design of co-culture experiment (A). Images taken with 10X objective (scale bars = 500 µm) at terminal differentiation following pre-exposure (day 9; B). Pparg (C), Cebpa (D), and Fabp4 (E) expression as assessed by qRT-PCR is suppressed in mouse MSCs “pre-exposed” to myeloma cells in vitro for 72-h prior to differentiation (first dotted line indicates the end of pre-exposure and start of differentiation), and levels are slightly suppressed throughout differentiation (second dotted line represents day 2 of adipogenic differentiation) (n=3); *p ≤ 0.05.

Figure 5

Adipogenic differentiation of 3T3-L1 preadipocytes is inhibited by MM.1S myeloma cells. 3T3-L1 adipocytes were assessed for Oil Red-O content either alone (control) or with exposure to MM.1S cells prior to differentiation process via indirect (+MM ID) or direct (+ MM D) co-culture (day 11). Lipid content is significantly reduced by MM.1S co-culture (indirect, MM ID; direct, MM D) during differentiation compared to 3T3-L1 cells on their own (CTRL); quantification of Oil Red-O staining (A). Expression of adipogenic transcripts Pparg (B) and Adipoq (C) are suppressed during differentiation in the presence of conditioned media from MM.1S cells; n=4. Significant differences in gene expression (p ≤ 0.05, FC |1.8|, red is upregulated, green is downregulated) in 3T3-L1 adipocytes exposed to myeloma cells for 48-h prior to differentiation as measured by microarray (D); control n=2, MM.1S n=3; *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 6

Cellular metabolism and key signaling pathways are altered in myeloma-associated mouse adipocytes. 3T3-L1 adipocytes exposed to MM.1S prior to differentiation have altered expression of genes involved with glycolysis (A), fatty acid metabolism (B), and mTORC signaling (C) as determined via gene set enrichment analysis (GSEA) analysis of 3T3-L1 microarray data. Expression of genes involved in lipid metabolism and encoding essential growth factors are downregulated in MM-3T3-L1 adipocytes (D) as visualized by string-db analysis of 3T3-L1 microarray data (p < 0.05, FC<−1.5).

Figure 7

Key signaling pathways are altered in myeloma-associated mouse 3T3-L1 adipocytes. Upregulated genes in MM-3T3-L1s are connected via the central node of Il6 as demonstrated graphically by string-db analysis of 3T3-L1 microarray data.

Figure 8

Exposure to myeloma cells prior to adipogenic differentiation induces SASP production in adipocyte precursors. 3T3-L1 cells exposed to MM.1S soluble factors during differentiation exhibit increased expression of: Il6, Cxcl1, and Cxcl12 after terminal differentiation (A). Mouse bone marrow MSCs were exposed to MM.1S cells in vitro for up to 72 h prior to adipogenic differentiation. Cells were harvested for RNA extraction after 24 (first dotted line), 48, and 72 (second dotted line; change to adipogenic media) hours of pre-exposure, and after the first treatment of adipogenic media (day 5). Cxcl1 (B), Cxcl2 (C), and Il6 (D) gene expression was quantified in mMSCs at each time point; n=3 per group, *p < 0.05, **p < 0.01. Reanalysis of previously published MSC gene expression data comparing bone marrow from normal donors (NBM) and myeloma donors (MM) examining the SASP gene cluster (E) data from Corre et al. 2007, Leukemia.; heatmap generated via Morpheus.

Figure 9

Direct co-culture of human mesenchymal stem cells (hMSCs) with myeloma cells reveals cell-line specific effects on lipid accumulation and cytokine production. Experimental design of co-culture experiment (A) where multiple myeloma (MM) cells (MM.1S, RPMI-8226, OPM-2) were added 2 days prior to the start of differentiation. Myeloma cells were allowed to persist during differentiation with adipogenic media. Fresh adipogenic media was incubator for 72 h prior to the collection of conditioned media. Conditioned media was collected and cells were fixed and stained on day 18 of differentiation, a few days prior to terminal differentiation. Adipocytes were fixed, stained (phalloidin=green, Oil Red-O=red, DAPI=blue), and imaged with a 10X objective after differentiation and co-culture with or without myeloma cells (B); images are from one hMSC donor, but are representative of n=3 donors. Lipid content was assessed by Oil Red-O staining, elution, and quantification (C). Cytokines were assessed in conditioned media by human cytokine array (R&D) (D); n=3 donors for each condition (control=naïve, +MM.1S, +OPM-2, +RPMI-8226). Significance was assessed via two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.